Growth differentiation factor 15 is a myomitokine governing systemic energy homeostasis.

Reduced mitochondrial electron transport chain activity promotes longevity and improves energy homeostasis via cell-autonomous and -non-autonomous factors in multiple model systems. This mitohormetic effect is thought to involve the mitochondrial unfolded protein response (UPRmt), an adaptive stress-response pathway activated by mitochondrial proteotoxic stress. Using mice with skeletal muscle-specific deficiency of Crif1 (muscle-specific knockout [MKO]), an integral protein of the large mitoribosomal subunit (39S), we identified growth differentiation factor 15 (GDF15) as a UPRmt-associated cell-non-autonomous myomitokine that regulates systemic energy homeostasis. MKO mice were protected against obesity and sensitized to insulin, an effect associated with elevated GDF15 secretion after UPRmt activation. In ob/ob mice, administration of recombinant GDF15 decreased body weight and improved insulin sensitivity, which was attributed to elevated oxidative metabolism and lipid mobilization in the liver, muscle, and adipose tissue. Thus, GDF15 is a potent mitohormetic signal that safeguards against the onset of obesity and insulin resistance.

Mitochondrial Protection by Exogenous Otx2 in Mouse Retinal Neurons.

OTX2 (orthodenticle homeobox 2) haplodeficiency causes diverse defects in mammalian visual systems ranging from retinal dysfunction to anophthalmia. We find that the retinal dystrophy of Otx2(+/GFP) heterozygous knockin mice is mainly due to the loss of bipolar cells and consequent deficits in retinal activity. Among bipolar cell types, OFF-cone bipolar subsets, which lack autonomous Otx2 gene expression but receive Otx2 proteins from photoreceptors, degenerate most rapidly in Otx2(+/GFP) mouse retinas, suggesting a neuroprotective effect of the imported Otx2 protein. In support of this hypothesis, retinal dystrophy in Otx2(+/GFP) mice is prevented by intraocular injection of Otx2 protein, which localizes to the mitochondria of bipolar cells and facilitates ATP synthesis as a part of mitochondrial ATP synthase complex. Taken together, our findings demonstrate a mitochondrial function for Otx2 and suggest a potential therapeutic application of OTX2 protein delivery in human retinal dystrophy.

Dysregulation of Parkin-mediated mitophagy in thyroid Hürthle cell tumors.

Abnormal accumulation of defective mitochondria is the hallmark of oncocytes, which are frequently observed in thyroid Hürthle cell lesions. Autophagy is an essential cellular catabolic mechanism for the degradation of dysfunctional organelles and has been implicated in several human diseases. It is yet unknown how autophagic turnover of defective mitochondria in Hürthle cell tumors is regulated. We characterized the expression patterns of molecular markers including Beclin1, LC3, PINK1 and Parkin, which are required for autophagy or mitophagy, in human oncocytic lesions of the thyroid. To undertake mechanistic studies, we investigated autophagy and mitophagy using XTC.UC1 cells, the only in vitro model of Hürthle cell tumors. Beclin1 and LC3 were highly expressed in oncocytes of Hürthle cell tumors. XTC.UC1 showed autophagic responses to starvation and rapamycin treatment, whereas they displayed ineffective activation of mitophagy, which is triggered by the coordinated action of PINK1 and Parkin in response to CCCP. This resulted in a decreased turnover of abnormal mitochondria. The mechanisms underlying defective mitophagy and mitochondrial turnover were investigated by genetic analysis of the PARK2 gene in XTC.UC1 and Hürthle cell tumor tissues. XTC.UC1 and several tumors harbored the V380L mutation, resulting in dysfunctional autoubiquitination and decreased E3 ligase activity. Consistently, oncocytes in Hürthle cell tumors displayed comparable expression of PINK1 but decreased Parkin expression in comparison to normal thyrocytes. The introduction of wild-type Parkin sensitized XTC.UC1 to death induced by CCCP. This study provides a possible etiological basis for oncocytic formation in heterogeneous Hürthle cell tumors through insufficient mitophagy leading to ineffective turnover of aberrant mitochondria caused by dysfunctional Parkin-mediated pathways of mitochondria quality control.

Disruption of CR6-interacting factor-1 (CRIF1) in mouse islet beta cells leads to mitochondrial diabetes with progressive beta cell failure.

AIM/HYPOTHESIS: Although mitochondrial oxidative phosphorylation (OxPhos) dysfunction is believed to be responsible for beta cell dysfunction in insulin resistance and mitochondrial diabetes, the mechanisms underlying progressive beta cell failure caused by defective mitochondrial OxPhos are largely unknown. METHODS: We examined the in vivo phenotypes of beta cell dysfunction in beta cell-specific Crif1 (also known as Gadd45gip1)-deficient mice. CR6-interacting factor-1 (CRIF1) is a mitochondrial protein essential for the synthesis and formation of the OxPhos complex in the inner mitochondrial membrane. RESULTS: Crif1(beta-/-) mice exhibited impaired glucose tolerance with defective insulin secretion as early as 4 weeks of age without defects in islet structure. At 11 weeks of age, Crif1(beta-/-) mice displayed characteristic ultrastructural mitochondrial abnormalities as well as severe glucose intolerance. Furthermore, islet area and insulin content was decreased by approximately 50% compared with wild-type mice. Treatment with the glucoregulatory drug exenatide, a glucagon-like peptide-1 (GLP-1) agonist, was not sufficient to preserve beta cell function in Crif1(beta-/-) mice. CONCLUSIONS/INTERPRETATION: Our results indicate that mitochondrial OxPhos dysfunction triggers progressive beta cell failure that is not halted by treatment with a GLP-1 agonist. The Crif1(beta-/-) mouse is a useful model for the study of beta cell failure caused by mitochondrial OxPhos dysfunction.

Thyroid dysfunction associated with follicular cell steatosis in obese male mice and humans.

Adult thyroid dysfunction is a common endocrine disorder associated with an increased risk of cardiovascular disease and mortality. A recent epidemiologic study revealed a link between obesity and increased prevalence of hypothyroidism. It is conceivable that excessive adiposity in obesity might lead to expansion of the interfollicular adipose (IFA) depot or steatosis in thyroid follicular cells (thyroid steatosis, TS). In this study, we investigated the morphological and functional changes in thyroid glands of obese humans and animal models, diet-induced obese (DIO), ob/ob, and db/db mice. Expanded IFA depot and TS were observed in obese patients. Furthermore, DIO mice showed increased expression of lipogenesis-regulation genes, such as sterol regulatory element binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor γ (PPARγ), acetyl coenzyme A carboxylase (ACC), and fatty acid synthetase (FASN) in the thyroid gland. Steatosis and ultrastructural changes, including distension of the endoplasmic reticulum (ER) and mitochondrial distortion in thyroid follicular cells, were uniformly observed in DIO mice and genetically obese mouse models, ob/ob and db/db mice. Obese mice displayed a variable degree of primary thyroid hypofunction, which was not corrected by PPARγ agonist administration. We propose that systemically increased adiposity is associated with characteristic IFA depots and TS and may cause or influence the development of primary thyroid failure.

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy.

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), ß (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism.

NAD(P)H: quinone oxidoreductase 1 and NRH:quinone oxidoreductase 2 polymorphisms in papillary thyroid microcarcinoma: correlation with phenotype.

PURPOSE: NAD(P)H:Quinone Oxidoreductase 1 (NQO1) C609T missense variant (NQO1*2) and 29 basepair (bp)-insertion/deletion (I29/D) polymorphism of the NRH:Quinone Oxidoreductase 2 (NQO2) gene promoter have been proposed as predictive and prognostic factors for cancer development and progression. The purpose of this study is to investigate the relationship between NQO1/NQO2 genotype and clinico-pathological features of papillary thyroid microcarcinoma (PTMC). MATERIALS AND METHODS: Genomic DNA was isolated from 243 patients; and clinical data were retrospectively analyzed. NQO1*2 and tri-allelic polymorphism of NQO2 were investigated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: PTMC with NQO1*2 frequently exhibited extra-thyroidal extension as compared to PTMC with wild-type NQO1 (p=0.039). There was a significant relationship between I29/I29 homozygosity of NQO2 and lymph node metastasis (p=0.042). Multivariate analysis showed that the I29/I29 genotype was associated with an increased risk of lymph node metastasis (OR, 2.24; 95% CI, 1.10-4.56; p=0.026). CONCLUSION: NQO1*2 and I29 allele of the NQO2 are associated with aggressive clinical phenotypes of PTMC, and the I29 allele represents a putative prognostic marker for PTMC.

Crif1 deficiency reduces adipose OXPHOS capacity and triggers inflammation and insulin resistance in mice.

Impaired mitochondrial oxidative phosphorylation (OXPHOS) has been proposed as an etiological mechanism underlying insulin resistance. However, the initiating organ of OXPHOS dysfunction during the development of systemic insulin resistance has yet to be identified. To determine whether adipose OXPHOS deficiency plays an etiological role in systemic insulin resistance, the metabolic phenotype of mice with OXPHOS-deficient adipose tissue was examined. Crif1 is a protein required for the intramitochondrial production of mtDNA-encoded OXPHOS subunits; therefore, Crif1 haploinsufficient deficiency in mice results in a mild, but specific, failure of OXPHOS capacity in vivo. Although adipose-specific Crif1-haploinsufficient mice showed normal growth and development, they became insulin-resistant. Crif1-silenced adipocytes showed higher expression of chemokines, the expression of which is dependent upon stress kinases and antioxidant. Accordingly, examination of adipose tissue from Crif1-haploinsufficient mice revealed increased secretion of MCP1 and TNFα, as well as marked infiltration by macrophages. These findings indicate that the OXPHOS status of adipose tissue determines its metabolic and inflammatory responses, and may cause systemic inflammation and insulin resistance.

Mitochondrial oxidative phosphorylation reserve is required for hormone- and PPARγ agonist-induced adipogenesis.

Adipocyte differentiation requires the coordinated activities of several nuclear transcription factors. Recently, mitochondria biogenesis was reported to occur during adipocyte differentiation and following treatment with thiazolidinediones in vitro and in vivo. Crif1 is a translational factor for mitochondrial DNA (mtDNA) and is important for transcription of the mitochondrial oxidative phosphorylation (OXPHOS) complex. To investigate the role of OXPHOS in adipogenesis, we analyzed adipocyte differentiation following disruption of Crif1 in vitro and in vivo. The adipose-specific Crif1 knockout mouse had a lower body weight and less fat mass than wild-type mice. Furthermore, adipocytes were smaller and had a dysplastic morphology in the adipose-specific Crif1 knockout mouse. 3T3-L1 adipocytes or adipose-derived stem cells (ADSCs) that lacked Crif1 expressed lower levels of mtDNA-encoded OXPHOS subunits, and adipocyte differentiation was disrupted. Rosiglitazone treatment did not induce adipogenesis or mitochondria biogenesis in Crif1 knockout ADSCs. These results show that mitochondrial OXPHOS and Crif1 are required for rosiglitazone- and hormone-induced adipogenesis.

CRIF1 is essential for the synthesis and insertion of oxidative phosphorylation polypeptides in the mammalian mitochondrial membrane.

Although substantial progress has been made in understanding the mechanisms underlying the expression of mtDNA-encoded polypeptides, the regulatory factors involved in mitoribosome-mediated synthesis and simultaneous insertion of mitochondrial oxidative phosphorylation (OXPHOS) polypeptides into the inner membrane of mitochondria are still unclear. In the present study, disruption of the mouse Crif1 gene, which encodes a mitochondrial protein, resulted in a profound deficiency in OXPHOS caused by the disappearance of OXPHOS subunits and complexes in vivo. CRIF1 was associated with large mitoribosomal subunits that were located close to the polypeptide exit tunnel, and the elimination of CRIF1 led to both aberrant synthesis and defective insertion of mtDNA-encoded nascent OXPHOS polypeptides into the inner membrane. CRIF1 interacted with nascent OXPHOS polypeptides and molecular chaperones, e.g., Tid1. Taken together, these results suggest that CRIF1 plays a critical role in the integration of OXPHOS polypeptides into the mitochondrial membrane in mammals.

Dual specificity phosphatase 6 as a predictor of invasiveness in papillary thyroid cancer.

OBJECTIVE: The genetic mutations causing the constitutive activation of MEK/ERK have been regarded as an initiating factor in papillary thyroid carcinoma (PTC). The ERK-specific dual specificity phosphatase 6 (DUSP6) is part of the ERK-dependent transcriptional output. Therefore, the coordinated regulation of the activities of ERK kinases and DUSP6 may need to be reestablished to make new balances in PTC. METHODS: To investigate the role of DUSP6 in the regulation of ERK1/2 (MAPK3/1)-dependent transcription, 42 benign neoplasms and 167 PTCs were retrospectively analyzed by immunohistochemistry with dideoxy sequencing to detect BRAF(V600E) mutation. RESULTS: The expressions of total ERK1/2, DUSP6, c-Fos (FOS), c-Myc (MYC), cyclin D1, and PCNA were markedly increased in PTC compared with those in benign neoplasms. However, phospho-ERK1/2 was detected in only eight (4.8%) cases out of 167 PTC samples. Unexpectedly, the staining intensity and nuclear localization of ERK1/2 were not affected by the presence or absence of the BRAF(V600E) mutation. However, the expressions of c-Fos and PCNA were elevated in BRAF(V600E)-positive PTC compared with those in BRAF(V600E)-negative PTC. Interestingly, the higher staining intensities of DUSP6 were associated with the level of total ERK1/2 expression (P=0.04) and with high-risk biological features such as age (P=0.05), tumor size (P=0.01), and extrathyroidal extension (linear by linear association, P=0.02). In addition, DUSP6 silencing significantly decreased the cell viability and migration rate of FRO cells. CONCLUSIONS: The coordinated upregulation of total ERK1/2 and its phosphatase, DUSP6, is related to bare detection of phospho-ERK1/2 in PTC regardless of BRAF(V)(600E) mutation status. A link between DUSP6 expression and high-risk features of PTC suggested that DUSP6 is an important independent factor affecting the signaling pathways in established PTC.

Aberrant l1 cell adhesion molecule affects tumor behavior and chemosensitivity in anaplastic thyroid carcinoma.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is one of the most invasive human cancers and has a poor prognosis. Molecular targets of ATC that determine its highly aggressive nature remain unidentified. This study investigated L1 cell adhesion molecule (L1CAM) expression and its role in tumorigenesis of ATCs. EXPERIMENTAL DESIGN: Expression of L1CAM in thyroid cancer was evaluated by immunohistochemical analyses of tumor samples from patients with thyroid cancer. We investigated the role of L1CAM in proliferation, migration, invasion, and chemoresistance using short hairpin RNA (shRNA) knockdown experiments in human ATC cell lines. Finally, we evaluated the role of L1CAM on tumorigenesis with ATC xenograft assay in a nude mouse model. RESULTS: L1CAM expression was not detectable in normal follicular epithelial cells of the thyroid or in differentiated thyroid carcinoma. In contrast, analysis of ATC samples showed specifically higher expression of L1CAM in the invasive area of the tumor. Specific knockdown of L1CAM in the ATC cell lines, FRO and 8505C, caused a significant decrease in the proliferative, migratory, and invasive capabilities of the cells. Suppression of L1CAM expression in ATC cell lines increased chemosensitivity to gemcitabine or paclitaxel. Finally, in an ATC xenograft model, depletion of L1CAM markedly reduced tumor growth and increased the survival of tumor-bearing mice. CONCLUSIONS: We report that L1CAM is highly expressed in the samples taken from patients with ATCs. L1CAM plays an important role in determining tumor behavior and chemosensitivity in cell lines derived from ATCs. Therefore, we suggest that L1CAM may be an important therapeutic target in patients with ATCs.

DJ-1 null dopaminergic neuronal cells exhibit defects in mitochondrial function and structure: involvement of mitochondrial complex I assembly.

DJ-1 is a Parkinson's disease-associated gene whose protein product has a protective role in cellular homeostasis by removing cytosolic reactive oxygen species and maintaining mitochondrial function. However, it is not clear how DJ-1 regulates mitochondrial function and why mitochondrial dysfunction is induced by DJ-1 deficiency. In a previous study we showed that DJ-1 null dopaminergic neuronal cells exhibit defective mitochondrial respiratory chain complex I activity. In the present article we investigated the role of DJ-1 in complex I formation by using blue native-polyacrylamide gel electrophoresis and 2-dimensional gel analysis to assess native complex status. On the basis of these experiments, we concluded that DJ-1 null cells have a defect in the assembly of complex I. Concomitant with abnormal complex I formation, DJ-1 null cells show defective supercomplex formation. It is known that aberrant formation of the supercomplex impairs the flow of electrons through the channels between respiratory chain complexes, resulting in mitochondrial dysfunction. We took two approaches to study these mitochondrial defects. The first approach assessed the structural defect by using both confocal microscopy with MitoTracker staining and electron microscopy. The second approach assessed the functional defect by measuring ATP production, O(2) consumption, and mitochondrial membrane potential. Finally, we showed that the assembly defect as well as the structural and functional abnormalities in DJ-1 null cells could be reversed by adenovirus-mediated overexpression of DJ-1, demonstrating the specificity of DJ-1 on these mitochondrial properties. These mitochondrial defects induced by DJ-1mutation may be a pathological mechanism for the degeneration of dopaminergic neurons in Parkinson's disease.

Cross-regulation between oncogenic BRAF(V600E) kinase and the MST1 pathway in papillary thyroid carcinoma.

BACKGROUND: The BRAF(V600E) mutation leading to constitutive signaling of MEK-ERK pathways causes papillary thyroid cancer (PTC). Ras association domain family 1A (RASSF1A), which is an important regulator of MST1 tumor suppressor pathways, is inactivated by hypermethylation of its promoter region in 20 to 32% of PTC. However, in PTC without RASSF1A methylation, the regulatory mechanisms of RASSF1A-MST1 pathways remain to be elucidated, and the functional cooperation or cross regulation between BRAF(V600E) and MST1,which activates Foxo3,has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: The negative regulators of the cell cycle, p21 and p27, are strongly induced by transcriptional activation of FoxO3 in BRAF(V600E) positive thyroid cancer cells. The FoxO3 transactivation is augmented by RASSF1A and the MST1 signaling pathway. Interestingly, introduction of BRAF(V600E)markedly abolished FoxO3 transactivation and resulted in the suppression of p21 and p27 expression. The suppression of FoxO3 transactivation by BRAF(V600E)is strongly increased by coexpression of MST1 but it is not observed in the cells in which MST1, but not MST2,is silenced. Mechanistically, BRAF(V600E)was able to bind to the C-terminal region of MST1 and resulted in the suppression of MST1 kinase activities. The induction of the G1-checkpoint CDK inhibitors, p21 and p27,by the RASSF1A-MST1-FoxO3 pathway facilitates cellular apoptosis, whereas addition of BRAF(V600E) inhibits the apoptotic processes through the inactivation of MST1. Transgenic induction of BRAF(V600E)in the thyroid gland results in cancers resembling human papillary thyroid cancers. The development of BRAF(V600E)transgenic mice with the MST1 knockout background showed that these mice had abundant foci of poorly differentiated carcinomas and large areas without follicular architecture or colloid formation. CONCLUSIONS/SIGNIFICANCE: The results of this study revealed that the oncogenic effect of BRAF(V600E) is associated with the inhibition of MST1 tumor suppressor pathways, and that the activity of RASSF1A-MST1-FoxO3 pathways determines the phenotypes of BRAF(V600E) tumors.

Antidiabetic and antiobesity effects of Ampkinone (6f), a novel small molecule activator of AMP-activated protein kinase.

Adenosine 5'-monophosphate (AMP) activated protein kinase (AMPK) has emerged as an attractive target molecule for the treatment of metabolic disorders, including obesity and type 2 diabetes. In this study, we identified a novel small molecule, ampkinone (6f), as an indirect AMPK activator, which was derived from the small molecule library constructed by diversity-oriented synthesis. Ampkinone stimulated the phosphorylation of AMPK via the indirect activation of AMPK in various cell lines. Ampkinone-mediated activation of AMPK required the activity of LKB1 and resulted in increased glucose uptake in muscle cells. In addition, ampkinone-treated DIO mice significantly reduced total body weight and overall fat mass. Histological examination and measurement of lipid parameters showed that ampkinone effectively improved metabolic abnormalities in the DIO mice model. Our results demonstrate that ampkinone, a small molecule with a privileged benzopyran substructure, has a potential as a new class of therapeutic agent for antidiabetic and antiobesity treatment via the indirect stimulation of AMPK.

Association of LETM1 and MRPL36 contributes to the regulation of mitochondrial ATP production and necrotic cell death.

Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is a mitochondrial inner membrane protein that was first identified in Wolf-Hirschhorn syndrome, and was deleted in nearly all patients with the syndrome. LETM1 encodes for the human homologue of yeast Mdm38p, which is a mitochondria-shaping protein of unclear function. Here, we describe LETM1-mediated regulation of mitochondrial ATP production and biogenesis. We show that LETM1 overexpression can induce necrotic cell death in HeLa cells, in which LETM1 reduces mitochondrial biogenesis and ATP production. LETM1 acts as an anchor protein and associates with mitochondrial ribosome protein L36. Adenovirus-mediated overexpression of LETM1 reduced mitochondrial mass and expression of many mitochondrial proteins. LETM1-mediated inhibition of mitochondrial biogenesis enhanced glycolytic ATP supply and activated protein kinase B activity and cell survival signaling. The expression levels of LETM1 were significantly increased in multiple human cancer tissues compared with normals. These data suggest that LETM1 serves as an anchor protein for complex formation with the mitochondrial ribosome and regulates mitochondrial biogenesis. The increased expression of LETM1 in human cancer suggests that dysregulation of LETM1 is a key feature of tumorigenesis.

Regulation of OPA1-mediated mitochondrial fusion by leucine zipper/EF-hand-containing transmembrane protein-1 plays a role in apoptosis.

Carboxyl-terminal modulator protein (CTMP) is a tumor suppressor-like binding partner of Protein kinase B (PKB/Akt) that negative regulates this kinase. In the course of our recent work, we identified that CTMP is consistently associated with leucine zipper/EF-hand-containing transmembrane-1 (LETM1). Here, we report that adenovirus-LETM1 increased the sensitivity of HeLa cells to apoptosis, induced by either staurosporine or actinomycin D. As shown previously, LETM1 localized to the inner mitochondrial membrane. Electron-microscopy analysis of adenovirus-LETM1 transduced cells revealed that mitochondrial cristae were swollen in these cells, a phenotype similar to that observed in optic atrophy type-1 (OPA1)-ablated cells. OPA1 cleavage was increased in LETM1-overexpressing cells, and this phenotype was reversed by overexpression of OPA1 variant-7, a cleavage resistant form of OPA1. Taken together, these data suggest that LETM1 is a novel binding partner for CTMP that may play an important role in mitochondrial fragmentation via OPA1-cleavage.